Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole...

Vesicular Exocytosis

“Neurotransmission and Catecholamines Release”

Christian Amatore

Ecole Normale Supérieure, Département de Chimie

UMR CNRS-ENS-UPMC 8640 "PASTEUR"

Paris - France

Adapted from: http://www.abcam/neuroscience/




Adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm

The Chromaffin Cell

Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.

Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.

Photographs: release of insulin by pancreatic -cells. Robert Kennedy. Private communication. (2002). Left sketch adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm

E.L. Ciolkowski, K.M. Maness, P.S. Cahill, R.M. Wightman, D.H. Evans, B. Fosset, C. Amatore. Anal. Chem., 66, 1994, 3611.

10 µm

Problems Associated with Ultrafast Electrochemistry

IC

IFItot = IF + IC

Problems associated with applying ultrafast electrochemical perturbations:

Ohmic Drop:

E(t) = ZF IF + RuItot(t)

Cell Time Constant:

cell = RuCd

IC

IFItot = IF + IC

Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant

IC

IFItot = IF + IC

Ru 1/r0

Cd r02

IC and IF r02

Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant

IC

IFItot = IF + IC

Ru Itot r0 0

Ru Cd r0 0

o For Planar Diffusion:

o For Any Diffusional Regime:

Compensation of Ohmic Drop and Time Constant

ZF IF = E(t) - RuItot)

IC = Cd(dE/dt) - RuCd(dItot/dt)

E(t) ZF IF IF Itot – Cd(dE/dt)

Ultramicroelectrode (measurement)

Living Cell

Micropipette(stimulation)

Release

Petri dish with PBS

10 µm

Principle of Electroanalytical Measurements at Single Cells

Preparation of Platinized Carbon Fiber Ultramicroelectrodes

o Sensitive detection of H2O2 ( "normal" [H2O2]cellular 10-9 to 10‑6 M )

o Sensitive detection of other expected species (NO°, etc.)

o Aerobic conditions ( [O2] 0,23 mM at 25° C )

o Analysis medium: PBS

o Microsensor dimensions: adapted to cell dimensions

o Real-time detection of biological events.

o Intrinsic Requirementso Intrinsic Requirements

10-12 µm 1-5 µm

glass cases

insulatingpolymer

platinizedsurfaces

5 µm 5 µm

Qav = 0.9 pC Nav = 2.7 106 molecules

10 µm

Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4

0.

I.

0.

III. IV.

Five Independent Physicochemical Stages Govern Exocytosis:

T.J. Schroeder, R. Borges, K. Pihel, C. Amatore, R.M. Wightman. Biophys. J., 70, 1996, 1061-1068.

0

20

40

60

0 40 80 120cu

rren

t /pA

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.

Full FusionFull FusionFusion PoreFusion PoreDockingDocking

Docking Occurs at Specifically Structured Areas in Cell Membrane:

Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.Sketchs adapted from: Y. Humeau, F. Doussau, N.J. Grant, B. Poulain, Biochim., 82, 2000, 427-446.

Docking Phase: Structure of SNAREs Protein Assembly

Blocking Docking by Altering SNAREs Assembling with Botulin:

Cells transfected through electroporation with modified plasmides / DNA. Secretion elicited 48 hrs later with Ca2+, 2.5 mM.

C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.

Importance of SNAREs Assembling:

GFP alone (contro l 1 ; n=26)

GFP / S nap25 W T (contro l 2 ; n=21)

GFP / B otu lin A (n=21)GFP / S nap25 L203 (n=19)

0

20

40

60

Cu

mu

late

d S

ecr

etio

n E

ven

ts

0 40

time (s)

Botulin + GFP

Cells transfected through electroporation with modified plasmides / DNA. Secretion elicited 48 hrs later with Ca2+, 2.5 mM.

C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.


0.

I.

0.

III. IV.



0

20

40

60

0 40 80 120cu

rren

t /pA

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.


Pore Formation: The Stalk Model

Regulating Exocytosis with Exogenous Bilipids

R R Wpore 22

.

C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

2R

Surface tension Edge tension


Control


Control

LPC

AA

LPCNO P O

O

OO

H OH

O

AACO2H

LPCNO P O

O

OO

H OH

O

AACO2H


1400

Time (s)

0 50 100 150 200 250 300

# C

umu

late

d e

vent

s

0

200

400

600

800

1000

1200

AA

Control

LPC

Control

AA

(4 Hz)

(2.5 Hz)

(1 Hz)



U≠

pre - fusion

full fusion

U≠

Time (s)

0 50 100 150 200 250 300

Cum

ulat

ed e

vent

s

0

200

400

600

800

1000

1200

1400

LPC

AA

Control (U≠)LPC = kBT ln( ) - 1 kBT2.44

(U≠)AA = kBT ln( ) + 2 kBT 2.4 1

k = k0 exp(-U≠/kBT)


poregranulegranulediskfoot RCnFDii 4

Rpore /nm ≈ 0.3 x ifoot /pA


Release Through Initial Fusion Pore:

n = 2F = 96 500 Cb< Dgranule > = 4.8 10-8 cm2s-1

< Cgranule > = 0.6 M

Release Through Initial Fusion Pore:

Rpore /nm ≈ 0.3 x ifoot /pA

Rpore = (1.5 ± 0.5) nm

(patch-clamp measurements (Neher, Fernandez, etc.): Rpore between 1 and 3 nm)

0

20

40

60

0 40 80 120

i foot

= 6 pA

rpore

= 1.8 nm

curr

ent /

pA

time /ms

0

10

20

30

0 50 100

i foot

= 4 pA

rpore

= 1.2 nm

time /ms

0

15

30

0 40 80 120

i foot

= 3 pA

rpore

= 0.9 nm

time /ms

How Full Fusion May Follow Pore Release ?

R RW cellvespore 2 )( 2 .


R RW cellvespore 2 )( 2

How Full Fusion May Follow Pore Release ?

.


Full Fusion: Driving Force = Granule Swelling upon Release

Concept based on de Gennes’ "Blob Theory« , see e.g.:J.L. Barrat, J.F. Joanny, in Adv. Chem. Phys. (I. Prigogine & S. Rice, eds.). Vol 44, pp. 37-33. Wiley NY, 1996.

Photographs adapted from Geoffrey Fox:www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html


0.

I.

0.

III. IV.



0

20

40

60

0 40 80 120cu

rren

t /pA

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.


Rate of full fusion: surface area increases

Diffusion: control by Dt/Rvesicle2

.


Full Fusion: Two Phenomena Govern Spike Shapes:

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

t / ms

I(t) / Ipeak

or a(t)

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

I(t)

a(t) a(t) a(t)

I(t)I(t)

Release elicited by 10s BaCl2, 2 mM, in Locke buffer with MgCl2, 0.7 mM.


Full Fusion: Two Phenomena Govern Spike Shapes:

Rate of full fusion: surface area increases

Diffusion: control by Dt/Rvesicle2

W. Almers et al., Nature 406, 2000, 849-854.

o Evanescent wave spectroscopy:

Full Fusion Kineticso Amperommetry:

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

t / ms

I(t) / Ipeak

or a(t)

0

1

0 50

0

0,05

0,1

0,15

0,2

0 1 2 3 4 5

I(t)

a(t) a(t) a(t)

I(t)I(t)

Area

Time (ms)

"Seeing" & "Measuring" :Fluorescence and Amperommetry

vesicle

cell

First Half of Full Fusion

R RW cellvesreleased 2 2 )(

)/( 4 dtdRRWreleased

o Energy released:(a)

o Dissipation of energy released:(b)

C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.(a) : Energy of a membrane pore: Taupin and de Gennes

(b) : Rate law for viscous dissipation: F. Brochard-Wyart & colls., PNAS, 96, 1999,10591-10596.

0

0.2

0.4

0.6

0.8

0 0.25 0.5 0.75 1

(R /

Rve

sicl

e )

t / t 80%

stcellvesreleased c RW 2 )(


C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.

First Half of Full Fusion:Dissipation of Cell and Vesicle Membrane High Tensions

vesicle

cell

0 cellvesicle

C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.

Second Half of Full Fusion: Dissipation of Line Tension Between Relaxed Membranes

R /

Rve

sicl

e

%66%98

%66

tt

tt

0.2

0.4

0.6

0.8

1

0 0.25 0.5 0.75 1

Testing Our Model

C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.


)/( 4 dtdRRWreleased vesicle

cell

C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-

154.

fast

large

Testing Our Model



cell

C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-

154.

fast slow

large

small

Testing Our Model



cell

fast

large

Reducing , viz. the Driving Force, by Refraining Swelling

Photographs adapted from Geoffrey Fox:www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html

vesves

ves PR

2

10 pA10 s

La 3+ 10mMinjection

Electrode in contact with the cell

Reducing by Lanthanides Ions:


Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.



Increasing , viz. the Membrane Viscosity



Control Hyperosmotic

Increasing with a Hyperosmotic Shock:

Increasing , viz. Membrane Viscosity, with Hyperosmotic Shock:

970

mO

sm

Q / pC


K.P. Troyer, R.M. Wightman, J. Biol. Chem., 277, 2002, 29101-29107.

Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.



Decreasing and Increasing by Cell Membrane Tension

Cell Membrane Tension Through a Hypoosmotic Shock

Control Hypoosmotic

excess

Cell Membrane Tension Through a Hypoosmotic Shock

0 50 100 150 200 250 300 3500

200

400

600

800

1000

Hypo.

Control

2.4 Hz

3.7 Hz

Time / s

# C

umu

late

d E

ven

ts

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Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole...

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